Butyryl-CoA dehydrogenase from Megasphaera elsdenii. Specificity of the catalytic reaction

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Abstract
The absorption coefficient of butyryl-CoA dehydrogenase from M. elsdenii at 450 nm is determined as 14.4 mM-1 .cntdot. cm-1 in the CoA-free form and 14.2 mM-1 .cntdot. cm-1 in the CoA-liganded form (both yellow). The latter value is considerably higher than the earlier published estimate. Phenazine ethosulfate offers great advantages over phenazine methosulfate as a coupling dye in the catalytic assay despite giving lower Vmax values (506 min-1 as compared with 1250 min-1 under the conditions used). The phenazine ethosulfate assay is used to establish a pH optimum of 8.05 for oxidation of 100 .mu.M-butyryl-CoA. The rates of oxidation of a range of straight-chain, branched-chain and alicyclic acyl thioesters are used to provide the following information. Only straight-chain acyl groups containing 4-6 C atoms are easily accommodated by the postulated hydrophobic pocket of the enzyme. C-3-substituted acyl-CoA thioesters are not oxidized at a significant rate, suggesting that the C-3 pro-S H atom of straight-chain substrates is partially exposed to the solvent. Acyl-CoA thioesters with substitutions at C-2 are oxidized, though at a lower rate rate than their straight-chain counterparts. This implies that the C-2 pro-S H atom of straight-chain substrates is partially exposed to the solvent. Saturated alicyclic carboxylic acyl-CoA thioesters with 4-7 C atoms in the ring are oxidized, with maximal activity for the cyclohexane derivative. This implies that optimal oxidation requires a true trans orientation of the 2 departing H atoms. The strain imposed by bound unsaturated alicyclic acyl thioesters strikingly perturbs the flavin visible-absorption spectrum, with the exception of the cyclohex-2-ene derivative, which forms a complex with similar spectral properties to those of the crotonyl-CoA complex. In the thiol moiety of thioester substrates the amide bond of N-acetylcysteamine is essential for binding and catalysis. The adenosine structure contributes substantially to strong binding, but is less important in determining the catalytic rate.